My goal is to identify the molecular and cellular mechanisms of Angle-Closure Glaucoma (ACG), a severe subset of glaucoma. In ACG, due to a combination of various anatomical and physiological factors, the iris is pushed forward causing physical blockage of the ocular drainage structure. This results in inefficient aqueous humor exits, thereby causing high intraocular pressure (IOP) and glaucoma. The mechanisms underlying ACG are largely unidentified. I have recently characterized a mutant mouse that recapitulates features of human Primary ACG including modestly decreased ocular size, a relatively larger lens and a narrow angle. The causal mutation is in a gene coding for a novel serine protease Prss56. Importantly, mutations in the same gene contribute to ACG in humans with reduced posterior segment (posterior microphthalmia). I will exploit this mouse model to resolve the mechanisms underlying ACG. I have three aims: Aim 1: The known mutations in mouse and human PRS556 are not predicted to disrupt the catalytic activity of this protease. Hence, it is unclear if the mutant PRSS56-mediated ACG is controlled by its inability to proteolytically cleave endogenous substrate or by gaining a new or enhanced activity. To address this, I will generate mice with a Prss56 conditional allele that can be selectively inactivated using the cre/loxP system to give rise to a catalytically inactive protease. I will ablte Prss56 and determine their impact on ACG relevant phenotypes, including its effect on ocular axial length, angle configuration and IOP. Aim 2: 2a. I will test the contribution of the retina i mediating the effect of the mutant PRSS56. The retina is a strong candidate in mediating mutant Prss56-induced ACG. Signals from the retina are known to play an important role in determining ocular axial length. Therefore, abnormal retinal PRSS56 can induce reduced ocular size (an important component of ACG). Alteration in ocular size has been linked to changes in scleral composition, which can further exacerbate ACG by impeding transcleral fluid flow. I will conditionally ablate Prss56 only in the retinal cells and assess their effect on ACG relevant phenotypes. 2b. My studies using Prss56 mutant mice suggest that postnatal developmental decrease in ocular size alone is insufficient to cause angle closure and high IOP. Alterations in adult ocular tissues must also participate in disease progression. To determine a role of stage-specific changes in ACG, I will use an inducible Cre to ablate Prss56 selectively from eyes at different ages and assess ACG related phenotypes. Aim 3: Identification of PRSS56 protease substrates is critical in understanding the molecular pathways contributing to ACG. I will employ two state-of-the-art approaches to identify PRSS56 substrates. First, use an open-reading frame (ORF)- phage display array to identify targets that are cleaved by PRSS56. Second, employ a proteome-wide strategy named terminal amine isotopic labeling of substrates (TAILS) to identify PRSS56 substrates. I will validate the in vivo specificity of these interactions using molecular approaches.